C-terminal protein tagging

ABSTRACT

In general, the invention features proteins having covalently bonded C-terminal puromycin tags and methods for their production.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of utility application, U.S.Ser. No. 09/614,264, filed Jul. 12, 2000 which claims the benefit ofprovisional application, U.S. S. No. 60/143,339, filed Jul. 12, 1999,now abandoned.

BACKGROUND OF THE INVENTION

[0002] In general, the invention relates to methods of labelingproteins.

[0003] Covalent conjugates of polypeptides with non-peptide “labels” or“tags” form a useful class of reagents in protein research. A conjugateis usually intended to retain the native properties of the protein whilegaining a new, non-native property due to the label. Biotinylation, forexample, permits proteins to be separated, quantified, or immobilized bymechanisms based on the strong interaction of biotin with avidin orstreptavidin (Bayer & Wilchek (1990) Protein Biotinylation, Meth.Enzymol. 184:138-160). Fluorescent or metal-chelating groups can also beintroduced to generate newly modified proteins.

[0004] It would be convenient to be able to introduce a non-proteinlabel as the protein is produced, but this is only sometimes feasible,for example, if the peptide can be produced by chemical synthesis, andis impractical when the protein is produced biologically. Generally, theconjugate must be formed by treating the peptide with a functionalgroup-specific reagent that contains the label. Moreover, unless thepeptide contains only one group attacked by the reagent, this proceduregenerally yields a mixture of products. This random form of labeling issometimes adequate, but it is often preferable to modify a protein at asingle specified site and to employ the modified product in purifiedform. For such cases, it would be valuable to have a method of directingthe modifying group to a single, preselected location. Such a preciselytargeted modification is termed site-directed protein tagging.

SUMMARY OF THE INVENTION

[0005] In general, the invention features a protein having a covalentlybonded puromycin tag, the tag being positioned at the C-terminal end ofthe protein.

[0006] In preferred embodiments, the tag is a small molecule (forexample, biotin); the tag is a detectable label (for example,fluorescein, rhodamine, or BODIPY, or derivatives thereof); the tag is afunctional group (for example, a functional group having a reactivityorthogonal to the reactivity of one of the protein's functional groups);the tag is a tether for attachment to a solid support (for example, acolumn, bead, or chip); the tag is one member of a specific bindingpair; the tag is a phenyl diboronic acid derivative; the puromycin tagfurther includes a nucleotide sequence positioned between the tag andthe puromycin; and the nucleotide sequence is between about 1-200nucleotides in length.

[0007] In a related aspect, the invention features a method forC-terminal protein tagging, involving (a) providing a nucleic acidsequence encoding the protein; (b) translating the sequence underconditions in which translation stalls at the 3′ end of the sequence,forming a stalled translation complex; and (c) contacting the stalledtranslation complex with a puromycin tag under conditions in which thepuromycin tag is covalently bonded to the C-terminus of the protein.

[0008] In preferred embodiments, the tag is attached to the 5′-hydroxygroup of puromycin; the tag is attached to the 5′-hydroxy group of thepuromycin through a phosphate group; the nucleic acid sequence encodingthe protein contains no stop codons; the translation step is carried outin the substantial absence of at least one translation release factor;the 3′-end of the nucleic acid sequence encoding the protein iscovalently linked to a DNA oligomer; the tag is a small molecule (forexample, biotin); the tag is a detectable label (for example,fluorescein, rhodamine, or BODIPY, or a derivative thereof); the tag isa functional group; the protein has a first functional group and the tagis a second functional group, wherein the first functional group has areactivity orthogonal to the reactivity of the second functional group;the tag is a tether for attachment to a solid support (for example, acolumn, bead, or chip); the tag is one member of a specific bindingpair; the tag is a phenyl diboronic acid derivative; the puromycin tagfurther includes a nucleotide sequence positioned between the tag andthe puromycin; and the nucleotide sequence is between about 1-200nucleotides in length.

[0009] By a “protein” is meant any two or more naturally occurring ormodified amino acids joined by one or more peptide bonds. “Protein,”“peptide,” and “polypeptide” are used interchangeably herein.

[0010] By a “puromycin tag” is meant puromycin having a covalentlybonded structural or functional moiety which is not native to thepuromycin molecule and which is chosen from the group consisting of adetectable label, a chemically reactive functional group, a smallmolecule, a protein or peptide, a peptoid, a naturally occurring ornon-naturally occurring polymer, a solid-phase bound tether, acarbohydrate, or a nucleic acid (preferably, of between about 1-200nucleotides) which does not encode the protein to which the puromycintag is itself covalently linked. By a “nucleic acid” is meant any two ormore covalently bonded, naturally occurring or modified nucleotides andincludes DNA, RNA, and PNA. Preferred puromycin-nucleic acid tagsinclude 5′-C-C-puromycin-3′.

[0011] By a “small molecule” is meant a molecule having a molecularweight of approximately 2000 Daltons or less, preferably, 1500 Daltonsor less, more preferably, 1000 Daltons or less, and, most preferably,500 Daltons or less.

[0012] By a “functional group” is meant any moiety of, or arrangement ofatoms in, a molecule which exhibit some chemical reactivity.

[0013] The present invention provides a number of advantages overcurrent chemical and enzymatic protein-tagging methods. For example, thetag is introduced in the final step of translation on the ribosome. Thismodification is advantageous because tagged proteins may be generated ina single preparative step. In addition, the tag is introduced intranslation buffer under conditions which enhance protein stability.Again, this provides for increased product yield and optimized proteinquality. In particular, although several schemes for N-terminal(Drijfhout et al. (1990) Anal. Biochem. 187: 349-354; Wetzel et al.(1990) Bioconjugate Chem. 1: 114-122) and C-terminal (Schwarz et al.(1990) Meth. Enzymol. 184: 160-162; Rose et al. (1989) Peptides 1988 (G.Jung and E. Bayer, eds.) pp. 274-276, Walter de Gruyter & Co., New York)tagging have been described, many of these methods involve a taggingstep that is carried out under conditions which disrupt proteinstructure. For example, modification at non-physiological pH andtemperature or in the presence of non-aqueous solvents or chemicals,followed by purification of the modified protein, disrupts folding thusleading to a non-functional product. In contrast, the present taggingmethod is performed under native conditions. Finally, in yet anotheradvantage, the present invention enables the introduction of a tagregioselectively at the C-terminus of a protein, facilitating theproduction of native proteins carrying desired C-terminal structural orfunctional elements in a simple and efficient way.

[0014] The C-terminally tagged polypeptides and proteins produced by themethods of the present invention may be used in any appropriatetechnique, for example, in any affinity purification method, proteindetection method (for example, using proteins having C-terminalfluorescein tags), structure function or protein dynamics analyses (forexample, using proteins having C-terminal reporter tags), pharmaceuticalanalyses (for example, using proteins having detectable C-terminal tagswhich allow for a determination of cellular protein uptake or cellularlocalization), or protein display technology (for example, using solidphase tags to generate protein arrays on microchips).

[0015] Other features and advantages will be apparent from the followingdetailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic illustration of the preparation of aC-terminally tagged protein according to the present invention.

[0017]FIG. 2 is a schematic illustration of an exemplary method for theattachment of a tag to a puromycin or oligonucleotide-puromycin(X_(n)-puromycin) derivative through a 5′-phosphodiester linkage.

[0018]FIG. 3 is a schematic illustration of an exemplary method for theattachment of tags to puromycin or X_(n)-puromycin with 5′-terminalmodified amino or thiol linkers.

[0019]FIG. 4 is a schematic illustration of anX_(n)-puromycin-5′-phosphate carrying a 5′-tethered biotin.

[0020]FIG. 5 is a schematic illustration of an X_(n)-puromycin5′-phosphate carrying both biotin and fluorescein groups.

[0021]FIG. 6 is a schematic illustration of the reversible attachment ofa protein-linked X_(n)-puromycin-5′-phosphate carrying a 5′-tetheredphenyl diboronic acid to a salicylhydroxamic acid derivative.

[0022]FIG. 7 is a schematic illustration of anX_(n)-puromycin-5′-phosphate carrying a 5′-ketone group.

[0023]FIG. 8 is a schematic illustration of an exemplary method for theattachment of a 5′-terminal amino X_(n)-puromycin to an N-hydroxysuccinimide activated agarose gel (AffiGel).

[0024]FIG. 9 is a schematic illustration of an exemplary method for theattachment of a 5′-terminal amino X_(n)-puromycin to anisothiocyanate-functionalized chip surface, for example, for the purposeof directed protein immobilization.

[0025]FIG. 10 is a schematic illustration of an X_(n)-puromycin5′-phosphate dimer linked through a polyethylene oxide chain.

[0026]FIG. 11 is a schematic illustration of an exemplary method for theproduction of a 1,2-aminothiol puromycin derivative.

[0027]FIG. 12 is a schematic illustration of anX_(n)-puromycin-5′-phosphate carrying a 5′-terminal hydrazide group.

DETAILED DESCRIPTION

[0028] The present invention makes use of puromycin, an antibiotic thatmimics the aminoacyl end of tRNA, as a vehicle to introduce a tag at theC-terminus of a protein. The puromycin acts as a translation inhibitorby entering the ribosomal A site and accepting the nascent protein as aresult of the peptidyl transferase activity of the ribosome (Monro &Marcker (1967) J. Mol. Biol. 25: 347-350; Monro & Vazquez (1967) J. Mol.Biol. 28: 161-165). The resulting peptidyl-puromycin molecule contains astable amide linkage between the peptide and the O-methyl tyrosineportion of the puromycin.

[0029] A desired tag is linked to the puromycin moiety in such a waythat binding as well as peptidyl acceptor functionality of the puromycinis only slightly decreased or is not decreased at all. Followingtranslation, binding of the tagged puromycin by the ribosome followed bypeptidyl transfer onto the primary amino group of puromycin yields thedesired C-terminally tagged protein. This technique is shownschematically in FIG. 1.

[0030] Different positions of puromycin can serve as anchor points forattachment of tags, although puromycin functional groups not involved inribosome binding represent preferred anchor points for attachment. Inone particular example, it has been shown that the 5′-hydroxymethylgroup of puromycin does not contribute to ribosome binding (Vince et al.(1981) J. Med. Chem. 24: 1511). In addition, nucleic acids carrying a3′-terminal puromycin have been shown to bind to the ribosome.Accordingly, the 5′-hydroxy group of puromycin is a preferred positionfor attachment of a desired tag (Szostak et al., WO 98/31700; Roberts &Szostak (1997) Proc. Natl. Acad. Sci. USA 94: 12297-12302).

[0031] The attachment of additional nucleotides (preferably, betweenabout 1-200 nucleotides) at the 5′-hydroxymethyl group of puromycin may,in some cases, further enhance the ability of puromycin to enter theribosomal A site and to act as an effective tRNA substitute. Noparticular sequence of nucleotides is required for this purpose. In thiscase, the desired tag is linked through the 5′-position of whateverterminal nucleotide is employed. Such derivatives are referred to hereinas X_(n)-puromycin derivatives.

[0032] The tag can be any non-native structural or functional element.Small molecules, natural products, non-natural polymers, and solid-phasebound tethers represent tags according to the invention. Examples ofpreferred small molecules include, without limitation, biotin andfluorescein or any other detectable label. Examples of natural productsinclude, without limitation, peptides, proteins, nucleic acids, andcarbohydrates. Peptoids are an example of a preferred non-naturalpolymer tag (Zuckermann et al., J. Am. Chem. Soc. (1992) 114: 10646).Alternatively, the tag may be any functional group. Examples of usefulfunctional groups include those with reactivities orthogonal to thereactivities of protein functional groups, for example, double bonds andketones. In another aspect of the invention, the tag may be a tetherlinked to a solid phase. Such tags enable the ready attachment ofpeptides and proteins to columns, beads, or chip surfaces.

[0033] Any appropriate type of ligation chemistry may be exploited toattach the tag to the puromycin moiety and, for example, to the5′-hydroxy group of the puromycin. In a preferred embodiment of theinvention, the puromycin tags are synthesized using standard solid phasetechniques, for example, as outlined in FIG. 2. Commercially availablephosphoramidites of biotin or fluorescein (Glen Research), for example,may be used to derivatize the 5′-terminus of puromycin orX_(n)-puromycin. These reactions may be carried out, for example, asdescribed in Oligonucleotide Synthesis: A Practical Approach, ed. Gait,M. J. (IRL, Oxford).

[0034] Using the synthetic scheme shown in FIG. 2, any number of5′-tagged puromycin derivatives or X_(n)-puromycin derivatives may bereadily produced. These derivatives include puromycin or X_(n)-puromycinlinked to small molecules, for example, X_(n)-puromycin-5′-phosphatecarrying a tethered biotin derivative (FIG. 4); such a puromycin orX_(n)-puromycin tag may be used to attach a C-terminal biotin label to aprotein, for example, for affinity purification. In a further example, apuromycin or X_(n)-puromycin tag may act as a bifunctional reagent byusing a puromycin or X_(n)-puromycin derivative which contains bothattachment and detection groups, for example, as shown in FIG. 5. Inthis example, an X_(n)-puromycin derivative is tethered through its5′-phosphate to both biotin and fluorescein moieties using standardoligonucleotide synthesis techniques. Attachment groups may include,without limitation, biotin, phenyl diboronic acid/salicylhydroxamicacid, 1,2-amino thiol, or ketone. Detection groups may include, withoutlimitation, fluorescein or derivatives thereof, rhodamine or derivativesthereof, or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene,Molecular Probes, Eugene, Oreg.) or derivatives thereof.

[0035] In a further example, a puromycin or X_(n)-puromycin 5′-phosphatecarrying a phenyl diboronic acid (PDBA) derivative may also be used tolabel the C-terminus of a peptide or protein for the purpose ofpurification or immobilization, as shown, for example, in FIG. 6. Thisreaction is carried out, for example, according to the manufacturer'sinstructions (Linx™ AP system, Invitrogen, Carlsbad, Calif.). In thisexample, the PDBA moiety interacts specifically with a salicylhydroxamicacid derivative to form a covalent complex (Linx™ AP system, Invitrogen,Carlsbad, Calif.). The interaction is reversible under certain pHconditions.

[0036] The scheme in FIG. 3 outlines the synthesis of puromycin orX_(n)-puromycin tags which include a terminal modification thatintroduces a terminal amino or thiol functionality into the puromycin orX_(n)-puromycin intermediate. These reactions are again carried out asdescribed above for FIG. 2. As illustrated in FIG. 7, these reactivemoieties are then used to introduce non-protein functional groups, forexample, a ketone, into the puromycin or X_(n)-puromycin tag.

[0037] The synthetic scheme illustrated in FIG. 3 may also be used tolink puromycin to a solid phase, as shown in FIG. 8. The solid phase maybe any appropriate solid support including, without limitation, anycolumn, plate, tube, bead, or chip. In the example illustrated in FIG.8, a 5′-terminal amino functionalized puromycin or X_(n)-puromycin isused to derivatize an N-hydroxysuccinimide-activated agarose support(for example, Affi-Gel 10 or 15; Biorad, Hercules, Calif.) to generatean immobilized puromycin or X_(n)-puromycin derivative where, in thisexample, the solid support is the tag. This step is carried outaccording to the manufacturer's instructions (Affi-Gel; Biorad,Hercules, Calif.). The puromycin or X_(n)-puromycin tethered to thesolid phase may then be utilized in the purification and/orimmobilization of peptides or proteins.

[0038] A puromycin or X_(n)-puromycin derivative with the appropriateterminal reactivity may also be used to derivatize a functionalized chipsurface. The resulting “puromycin chip” may then be utilized for thedirect attachment of peptides or proteins on the chip surface uponcontact with stalled ribosome complexes, as illustrated in FIG. 9. Inthis example, a 5′-amino terminated X_(n)-puromycin derivative is usedto functionalize a chip surface premodified with isothiocyanate groups(as described, for example, in Kuimelis et al., U.S. Ser. No.09/282,734, entitled Addressable Protein Arrays, filed Mar. 31, 1999;and Kuimelis et al., WO 99/51773). The tethered puromycin then directsthe attachment of proteins through their C-terminus upon reaction withstalled ribosome complexes.

[0039] In yet another embodiment, puromycin may be linked to polymers.In one particular example, puromycin may be attached to anoligonucleotide using previously described methods (Szostak et al., WO98/31700; Szostak et al., U.S. Ser. No. 09/247,190 (1999); Roberts &Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302) for thepurpose of sequence-specific hybridization to a solid phase.

[0040] An appropriate puromycin or X_(n)-puromycin tag may also be usedfor the preparation of protein-protein conjugates. In one example, apuromycin or X_(n)-puromycin dimer (FIG. 10) may be added to stalledribosome complexes to generate protein homodimers. Puromycin orX_(n)-puromycin dimers are prepared as described above for FIG. 2 usinga second puromycin or puromycin derivative as a tag.

[0041] In an alternative synthetic scheme, protein-polymer conjugatesmay be prepared using a step-wise approach. In the first step, apuromycin or X_(n)-puromycin derivative carrying a 5′-cysteinol moietyis used to introduce a 1,2-aminothiol reactive group into a protein. Anexample of the preparation of this modified protein is outlined in FIG.11. In this example, a puromycin or X_(n)-puromycin derivative carryinga 5′-terminal thiol moiety is alkylated with a suitable protectedN-terminal cysteinyl peptide carrying a thiol-reactive maleimide groupat its C-terminus (as described, for example, in Boeckler et al. (1998)Bioorg. Med. Chem. Lett. 8:2055-2058). In the second step, polymers (forexample, proteins, nucleic acids, or unnatural polymers) or a solidphase (for example, a column, plate, tube, bead, or chip) carrying athiolester group are linked to the 1,2-aminothiol-tagged protein underphysiological conditions using an orthogonal ligation strategy, asdescribed, for example, in Methods for the Preparation of NucleicAcid-Protein Conjugates, Dawson et al. (1994) Science 266: 776; Ni etal. (1998) J. Am. Chem. Soc. 120: 1645; and McPherson et al. (1999) Syn.Lett. S1: 978-980.

[0042] Alternatively, a puromycin or X_(n)-puromycin derivative carryinga 5′-terminal hydrazide group, as shown in FIG. 12, may be used tointroduce a C-terminal hydrazide nucleophile into the protein (Lohse etal., DNA-Protein Fusions and Uses Thereof, U.S. S. No. 60/110,549; U.S.Ser. No. 09/453,190 (1999); and WO 00/32823). Reaction of the hydrazidewith a carbohydrate aldehyde or ketone group under physiologicalconditions may be used to generate protein-carbohydrate conjugates (asdescribed, for example, in Gahmberg & Tolvanen (1994) Meth. Enzymol 230:32-44).

[0043] To maximize the yield of the C-terminally tagged product, the tagis preferably attached to the full-length peptide or protein followingtranslation of the open reading frame. This can be achieved by stallingthe ribosome as an mRNA-ribosome-peptidyl complex after translation ofthe coding sequence. Ribosome stalling at the 3′-end of the open readingframe may be accomplished by any of a number of different methods. Inone preferred approach, the message is engineered to be devoid of stopcodons. As a result, release factors cannot bind, and the ribosomestalls (see, for example, Hanes & Plueckthun (1997) Proc. Natl. Acad.Sci. USA 94: 4937-4942). In another preferred approach, a DNA oligomermay be linked to the end of the message causing the ribosome to pause;this technique is described in Szostak et al., WO 98/31700; Szostak etal., U.S. Ser. No. 09/247,190 (1999); and Roberts & Szostak (1997) Proc.Natl. Acad. Sci. USA 94: 12297-12302). Alternatively, an in vitrotranslation lysate may be utilized which is devoid of release factors,as described in Lipovsek et al., Methods for Optimizing CellularRNA-Protein Fusion Formation, U.S. S. No. 60/096,818; U.S. Ser. No.09/374,962 (1999); and WO 00/09737.

[0044] The yield of the reaction of tagged puromycin with stalledribosomes depends on the Kd of the puromycin derivative for the peptidyltransferase site, the concentration of tagged puromycin, and reactionconditions like buffer, temperature, and time. For example, in apreferred approach, the synthetically prepared puromycin derivative maybe incubated under conditions known to stabilize stalled ribosomes (see,for example, Hanes & Plueckthun (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942). The concentration of tagged puromycin supplied to thestalled ribosomes should preferably be above the Kd of tagged puromycinfor the ribosomal peptidyl transferase. Concentrations of 5′-taggedpuromycin derivatives in the low mM range (or even the low μM range)allow efficient incorporation of the tag, considering the Kd ofunmodifed puromycin is in the micromolar range (see, for example, Pestka(1974) Meth. Enzymol. 30: 479-488; Vince et al. (1986) J. Med. Chem. 29:2400-2403).

[0045] Following ribosome-catalyzed peptidyl transfer onto boundpuromycin, the tagged protein may be released by addition of washingbuffer containing EDTA (see, for example, Hanes & Plueckthun (1997)Proc. Natl. Acad. Sci. USA, vol. 94, pp. 4937-4942). If desired, thetagged protein may then be purified using any appropriate biochemicalpurification protocol. In a preferred technique, the tag itself may beused to isolate the protein, for example, by affinity chromatography.Simple washing procedures may also be utilized if the puromycin istethered to a solid phase; in this approach, the tagged protein isretained on the solid support and impurities removed in the washsolution.

[0046] Experimental Results

[0047] The myc epitope was chosen as an example to highlight the generaltagging strategy described above. Myc dsDNA was generated by standardmethods of PCR to include a 5′-T7 promoter for in vitro synthesis ofmRNA using T7 polymerase and a deletion mutant of the tobacco mosaicvirus 5′-UTR to induce efficient translation initiation in rabbitreticulocyte lysates. The 3′-end of the myc construct was devoid of stopcodons to prevent protein release from the ribosome.

[0048] Transcription of the myc PCR product (using the MegaSHORTscriptkit, Ambion) gave large quantities of RNA using T7 RNA polymerase.Purified RNA was then subjected to a splinted ligation reaction with a5′-phosphorylated dA₃₀ oligonucleotide catalyzed by T4 DNA ligase. The3′-dA₃₀ region facilitates ribosomal stalling and thereby increases theproportion of RNA-ribosome-protein complexes available for labeling. Thepurified myc-dA₃₀ construct was then translated in rabbit reticulocytelysate (Ambion) with ³⁵S-methionine and stalled under high saltconditions (500 mM KCl, 20 mM MgCl₂).

[0049] Biotin-TEG-dCdC-puromycin (130 μM) was then added to the stalledtranslation reaction and labeling was allowed to take place.Biotin-TEG-dCdCdA was used as a control. The radiolabled myc peptide wasthen isolated by immunoprecipitation with an anti-myc monoclonalantibody (Chemicon) and protein A sepharose (Sigma). Control andC-terminally-labeled peptide were then applied to a microscope slideprefunctionalized with NeutrAvidin™ (Pierce). Visualization byphosphorimaging revealed the biotin-dependent immobilization of mycepitope to the chip surface; no significant label was associated withthe negative control.

[0050] In a separate set of experiments, similar tagging of thefibronectin type III domain was also carried out to demonstrate theutility of the present tagging strategy for the immobilization ofproteins with well-defined structural folds. Fibronectin is a largemulti-domain protein that plays a fundamental role in cell-cellinteractions and extracellular matrix formation. The repeating domainsdisplay immunoglobulin-like features that are widely involved inmammalian molecular recognition. A DNA construct of the tenth repeat ofthe human fibronectin type III domain (8 kDa, 10Fn3) was created thatincluded a region encoding an N-terminal His₆ tag in addition to 5′-T7promoter and TMV UTR regions.

[0051] Transcription (MegaSCRIPT, Ambion) of the ¹⁰Fn3 PCR product gavelarge amounts of RNA for subsequent enzymatic ligation to a dA₃₀oligonucleotide. Translation in rabbit reticulocyte lysate, stalling,and labeling with biotin-TEG-dCdC-puromycin were performed as outlinedin the myc experiment. ¹⁰Fn3 protein was isolated from excessbiotinylated puromycin analogue by affinity chromatography on a Co²⁺-NTAcolumn (Talon™, Clontech). Application of ¹⁰Fn3 to a chip surfaceprespotted with NeutrAvidin™ protein and phosphorimaging analysisrevealed the presence of radiolabeled ¹⁰Fn3.

[0052] The above experiments demonstrated that puromycin-mediated invitro attachment of labels to the C-termini of peptides or proteins is apowerful technique for the regiospecific introduction of non-naturalfunctionality into biomolecules. The puromycin analogues can besynthesized using standard oligonucleotide chemistry to include, forexample, fluorophores, spin labels, purification handles, or acombination thereof. One distinct advantage of this approach is thatlabeling is performed under in vitro conditions that are compatible withmaintaining the biological activity of the protein. Moreover, thistagging approach is amenable to a high throughput format of proteinlabeling for screening for biomolecules of therapeutic interest.

Other Embodiments

[0053] Other embodiments are within the claims.

[0054] All publications, patents, and patent applications mentionedherein are hereby incorporated by reference.

What is claimed is:
 1. A protein having a covalently bonded puromycintag, said tag being positioned at the C-terminal end of said protein. 2.The protein of claim 1, wherein said tag is a small molecule.
 3. Theprotein of claim 2, wherein said small molecule is biotin.
 4. Theprotein of claim 1, wherein said tag is a detectable label.
 5. Theprotein of claim 4, wherein said detectable label is fluorescein,rhodamine, or BODIPY, or derivatives thereof.
 6. The protein of claim 1,wherein said tag is a functional group.
 7. The protein of claim 1,wherein said protein has a first functional group and said tag is asecond functional group and wherein said first functional group has areactivity orthogonal to the reactivity of said second functional group.8. The protein of claim 1, wherein said tag is a tether for attachmentto a solid support.
 9. The protein of claim 8, wherein said solidsupport is a column, bead, or chip.
 10. The protein of claim 1, whereinsaid tag is one member of a specific binding pair.
 11. The protein ofclaim 10, wherein said tag is a phenyl diboronic acid derivative. 12.The protein of claim 1, wherein said puromycin tag further comprises anucleotide sequence positioned between said tag and said puromycin. 13.The protein of claim 12, wherein said nucleotide sequence is betweenabout 1-200 nucleotides in length.
 14. A method for C-terminal proteintagging, comprising (a) providing a nucleic acid sequence encoding saidprotein; (b) translating said sequence under conditions in whichtranslation stalls at the 3′ end of said sequence, forming a stalledtranslation complex; and (c) contacting said stalled translation complexwith a puromycin tag under conditions in which said puromycin tag iscovalently bonded to the C-terminus of said protein.
 15. The method ofclaim 14, wherein said tag is attached to the 5′-hydroxy group of saidpuromycin.
 16. The method of claim 15, wherein said tag is attached tothe 5′-hydroxy group of said puromycin through a phosphate group. 17.The method of claim 14, wherein said nucleic acid sequence encoding saidprotein contains no stop codons.
 18. The method of claim 14, whereinsaid translation step is carried out in the substantial absence of atleast one translation release factor.
 19. The method of claim 14,wherein the 3′-end of said nucleic acid sequence encoding said proteinis covalently linked to a DNA oligomer.
 20. The method of claim 14,wherein said tag is a small molecule.
 21. The method of claim 20,wherein said small molecule is biotin.
 22. The method of claim 14,wherein said tag is a detectable label.
 23. The method of claim 22,wherein said detectable label is fluorescein, rhodamine, or BODIPY, or aderivative thereof.
 24. The method of claim 14, wherein said tag is afunctional group.
 25. The method of claim 14, wherein said protein has afirst functional group and said tag is a second functional group andwherein said first functional group has a reactivity orthogonal to thereactivity of said second functional group.
 26. The method of claim 14,wherein said tag is a tether for attachment to a solid support.
 27. Themethod of claim 26, wherein said solid support is a column, bead, orchip.
 28. The method of claim 14, wherein said tag is one member of aspecific binding pair.
 29. The method of claim 28, wherein said tag is aphenyl diboronic acid derivative.
 30. The method of claim 14, whereinsaid puromycin tag further comprises a nucleotide sequence positionedbetween said tag and said puromycin.
 31. The method of claim 30, whereinsaid nucleotide sequence is between about 1-200 nucleotides in length.